Biology subjects

Weitzman, M. D.

Publications and source records attributed to Weitzman, M. D..

13 recordsLinked to original sources

bioRxiv2026

Virus-Driven Proximity Proteomics Identifies p400 as a Restriction Factor Poised on Incoming Viral Genomes

Immediate-early viral proteins must rapidly counteract host defenses directed at incoming viral genomes, yet the protein interaction networks that mediate this remain unresolved. This gap persists largely because immediate-early proteins are expressed at low abundance during this stage of infection, making them challenging to investigate with standard approaches. Here, we present a virus-driven proximity proteomics framework to investigate immediate-early phase virus-host interactions in an authentic infection context. An isogenic P2A control virus untethers a miniTurbo biotin ligase from the viral protein under investigation, effectively modeling bait abundance and background changes from a matched infection context. Continuous biotin labeling from the earliest hours of infection further amplifies detection of transient, low-abundance interactions characteristic of this phase. Using the adenovirus E1A hub protein as a benchmark, we recovered the majority of known E1A interactors and identified over 150 high-confidence interactions. Applying this strategy to the immediate-early phase, we resolved the E1A interactome and identified the p400 chromatin remodeling complex as its dominant target. We show p400 associates with incoming viral genomes, represses gene expression, and regulates persistent infection. These data identify p400 as a poised host restriction factor and establish a temporally resolved proximity proteomics strategy transferable to other immediate-early viral proteins.

microbiology↗

bioRxiv2026

HSV-1 hijacks the DNA repair protein RAD51 at gene promoters to drive viral transcription

Productive infection by Herpes Simplex Virus type 1 (HSV-1) requires initiation of efficient viral gene expression. Upon nuclear entry, HSV-1 genomes are associated with several cellular factors, including DNA repair proteins. It is unclear how these cellular factors impact viral processes at early stages of infection. In this study, we investigate the role of RAD51, a core homologous recombination (HR) factor. We measured nascent viral transcription and show that RAD51 plays a pro-viral role in infection by promoting immediate-early viral gene expression. We demonstrate that RAD51 binds GC-rich gene promoter of ICP4 and directly promotes gene expression. We also reveal a previously unknown interaction between RAD51 and ADNP, a subunit of the ChAHP complex, known for its role in transcription regulation. We propose a model where RAD51 binds incoming genomes at promoter regions regulating the genome landscape and allowing for efficient transcription initiation.

cell biology↗

bioRxiv2026

Differential assembly of RNP granules via activation of distinct dsRNA sensors by adenovirus mutants

Recognition of dsRNA triggers antiviral defense mediated by PKR and OAS3/RNase L pathways through translational arrest and RNA decay. This is accompanied by assembly of distinct cytoplasmic ribonucleoprotein (RNP) condensates termed stress granules (SGs) and RNase L-dependent bodies (RLBs). Here we show that adenovirus infection differentially modulates dsRNA sensors and RNP granule assembly. Infection with splicing-defective {Delta}E4 mutant leads to dsRNA accumulation and activation of both PKR and OAS3/RNase L, promoting formation of RLB-like granules. In contrast, mutants lacking virus-associated (VA) RNAs trigger PKR activation and assembly of SGs despite absence of detectable dsRNA. Proteomic analysis revealed distinct protein compositions of canonical SGs and RLBs, which were reflected in virus-induced granules. While {Delta}VA-induced granules were PKR-dependent, {Delta}E4 mutants induced RLB-like granules independently of PKR and RNase L. In these cells, granule assembly coincided with translational arrest independent of eIF2 phosphorylation, indicating additional pathways linking nuclear dsRNA sensing to translational control and RNP granule assembly during viral infection. These findings provide novel insights into how distinct dsRNA sensors modulate translation and RNP condensates in response to stress.

cell biology↗

bioRxiv2023

The SMC5/6 complex is required for maintenance of genome integrity upon APOBEC3A-mediated replication stress

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

molecular biology↗

bioRxiv2023

Double-Strand Break Repair Pathways Differentially Affect Processing and Transduction by Dual AAV Vectors

Recombinant adeno-associated viral vectors (rAAV) are a powerful tool for gene delivery but have a limited DNA carrying capacity. Efforts to expand this genetic payload have focused on engineering the vector components, such as dual trans-splicing vectors which double the delivery size by exploiting the natural concatenation of rAAV genomes in host nuclei. We hypothesized that inefficient dual vector transduction could be improved by modulating host factors which affect concatenation. Since factors mediating concatenation are not well defined, we performed a genome-wide screen to identify host cell regulators. We discovered that Homologous Recombination (HR) is inhibitory to dual vector transduction. We demonstrate that depletion or inhibition of HR factors BRCA1 and Rad51 significantly increase reconstitution of a large split transgene by increasing both concatenation and expression from rAAVs. Our results define new roles for DNA damage repair in rAAV transduction and highlight the potential for pharmacological intervention to increase genetic payload of rAAV vectors.

molecular biology↗

bioRxiv2023

METTL3 is essential for small intestinal epithelial proliferation via regulation of growth factor signaling including KRAS

Intestinal epithelial transit amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite their critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit amplifying cell function. We report that the RNA methyltransferase, METTL3, is required for survival of transit amplifying cells in the murine small intestine. Transit amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Ribosome profiling and sequencing of methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of unique methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation confirmed a novel relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine, with important implications for both homeostatic tissue renewal and epithelial regeneration.

cell biology↗

bioRxiv2020

Viral-mediated ubiquitination impacts interactions of host proteins with viral RNA and promotes viral RNA processing

Viruses promote infection by hijacking host ubiquitin machinery to counteract or redirect cellular processes. Adenovirus encodes two early proteins, E1B55K and E4orf6, that together co-opt a cellular ubiquitin ligase complex to overcome host defenses and promote virus production. Adenovirus mutants lacking E1B55K or E4orf6 display defects in viral RNA processing and protein production, but previously identified substrates of the redirected ligase do not explain these phenotypes. Here we used a quantitative proteomics approach to identify substrates of E1B55K/E4orf6-mediated ubiquitination that facilitate RNA processing. While all currently known cellular substrates of E1B55K/E4orf6 are degraded by the proteasome, we uncovered RNA-binding proteins (RBPs) as high-confidence substrates which are not decreased in overall abundance. We focused on two RBPs, RALY and hnRNP-C, which we confirm are ubiquitinated without degradation. Knockdown of RALY and hnRNP-C increased levels of viral RNA splicing, protein abundance, and progeny production during infection with E1B55K-deleted virus. Furthermore, infection with virus deleted for E1B55K resulted in increased interaction of hnRNP-C with viral RNA, and attenuation of viral RNA processing. These data suggest viral-mediated ubiquitination of RALY and hnRNP-C relieves a restriction on viral RNA processing, revealing an unexpected role for non-degradative ubiquitination in manipulation of cellular processes during virus infection.

microbiology↗

bioRxiv2020

The Viral Polymerase Complex Mediates the Interaction of vRNPs with Recycling Endosomes During SeV Assembly

Paramyxoviruses are negative sense single-stranded RNA viruses that comprise many important human and animal pathogens, including human parainfluenza viruses. These viruses bud from the plasma membrane of infected cells after the viral ribonucleoprotein complex (vRNP) is transported from the cytoplasm to the cell membrane via Rab11a-marked recycling endosomes. The viral proteins that are critical for mediating this important initial step in viral assembly are unknown. Here we use the model paramyxovirus, murine parainfluenza virus 1, or Sendai virus (SeV), to investigate the roles of viral proteins in Rab11a-driven virion assembly. We previously reported that infection with SeV containing high levels of copy-back defective viral genomes (DVGs) generates heterogenous populations of cells. Cells enriched in full-length virus produce viral particles containing standard or defective viral genomes, while cells enriched in DVGs do not, despite high levels of defective viral genome replication. Here we take advantage of this heterogenous cell phenotype to identify proteins that mediate interaction of vRNPs with Rab11a. We examine the role of matrix protein and nucleoprotein and determine that they are not sufficient to drive interaction of vRNPs with recycling endosomes. Using a combination of mass spectrometry and comparative protein abundance and localization in DVG- and FL-high cells, we identify viral polymerase complex components L and, specifically, its cofactor C proteins as interactors with Rab11a. We find that accumulation of these proteins within the cell is the defining feature that differentiates cells that proceed to viral egress from cells which remain in replication phases. IMPORTANCEParamyxoviruses are a family of viruses that include a number of pathogens with significant burdens on human health. Particularly, human parainfluenza viruses are an important cause of pneumonia and bronchiolitis in children for which there are no vaccines or direct acting antivirals. These cytoplasmic replicating viruses bud from the plasma membrane and coopt cellular endosomal recycling pathways to traffic viral ribonucleoprotein complexes from the cytoplasm to the membrane of infected cells. The viral proteins required for viral engagement with the recycling endosome pathway are still not known. Here we use the model paramyxovirus Sendai virus, or murine parainfluenza virus 1, to investigate the role of viral proteins in this initial step of viral assembly. We find that viral polymerase components large protein L and accessory C proteins are necessary for engagement with recycling endosomes. These findings are important in identifying viral proteins as potential targets for development of antivirals.

microbiology↗

bioRxiv2019

Novel splicing and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts

Adenovirus is a common human pathogen that relies on host cell processes for production and processing of viral RNA. Although adenoviral promoters, splice junctions, and cleavage and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and cleavage and polyadenylation sites across the adenovirus genome. The canonical viral early and late RNA cassettes were confirmed, but analysis of splice junctions within long RNA reads revealed an additional 20 novel viral transcripts. These RNAs include seven new splice junctions which lead to expression of canonical open reading frames (ORF), as well as 13 transcripts encoding for messages that potentially alter protein functions through truncations or the fusion of canonical ORFs. In addition, we also detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking separate gene transcription units. Our work highlights how long-read sequencing technologies can reveal further complexity within viral transcriptomes.

microbiology↗

bioRxiv2019

Direct RNA sequencing reveals m6A modifications on adenovirus RNA are necessary for efficient splicing

Adenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of N6-methyladenosine (m6A) to mRNAs. While m6A-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m6A mapping using short-read sequencing, we profiled m6A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m6A detection. Although both early and late viral transcripts contain m6A, depletion of m6A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m6A discovery within individual transcripts at nucleotide resolution, and highlight the role of m6A in regulating splicing of a viral pathogen.

molecular biology↗

bioRxiv2019

Impaired tumor death receptor signaling drives resistance to CAR T cell therapy

Cellular immunotherapy using T cells engineered to express chimeric antigen receptors targeting CD19 (CART19) leads to long-term remission in patients with B-cell malignancies1-5. Unfortunately, a significant fraction of patients demonstrate primary resistance to CART19 or experience relapse after achieving remission. Beyond loss of target antigen, the molecular pathways governing CART19 failure are unknown. Here we demonstrate that death receptors, cell surface signaling molecules that induce target cell apoptosis, are key mediators of leukemic resistance and CART19 failure. Using a functional CRISPR/Cas9-based genome-wide knockout screen6 in B-cell acute lymphoblastic leukemia (ALL), we identified the death receptor signaling pathway as a central regulator of sensitivity to CART19-induced cell death. In the absence of the pro-apoptotic death receptor signaling molecules BID or FADD, ALL cells were resistant to CART19 cytotoxicity, resulting in rapid disease progression in mice. We found that this initial resistance to cytotoxicity led to persistence of tumor cells, which drove the development of T cell dysfunction that further compromised anti-tumor immunity and permitted tumor outgrowth. We validated these findings using clinical samples collected from patients with ALL treated with CD19-targeted CAR T cells and found that expression of pro-apoptotic death receptor pathway genes in pre-treatment tumor samples correlated with CAR T cell expansion and persistence, as well as patient response and overall survival. Our findings indicate that tumor-intrinsic death receptor signaling directly contributes to CAR T cell failure.

immunology↗

bioRxiv2018

Recombination between co-infecting herpesviruses occurs where replication compartments coales

Homologous recombination (HR) is considered a major driving force of evolution since it generates and expands genetic diversity. Evidence of HR between co-infecting herpesvirus DNA genomes can be found frequently, both in vitro and in clinical isolates. Each herpes simplex virus type 1 (HSV-1) replication compartment (RC) derives from a single incoming genome and maintains a specific territory within the nucleus. This raises intriguing questions about where and when co-infecting viral genomes interact. To study the spatiotemporal requirements for inter-genomic recombination, we developed an assay with dual-color fluorescence in situ hybridization which enables detection of HR between different pairs of co-infecting HSV-1 genomes. Our results revealed that when viral RCs enlarge towards each other, there is detectable overlap between territories of genomes from each virus. Infection with paired viruses that allow visualization of HR correlates with increased overlap of RCs. Further, inhibition of RC movement reduces the rate of HR events among co-infecting viruses. Taken together, these findings suggest that inter-genomic HR events take place during replication of HSV-1 DNA and are mainly confined to the periphery of RCs when they coalesce. Our observations have implications on understanding the recombination restrictions of other DNA viruses and cellular DNA.

microbiology↗

bioRxiv2018

Genotypic and phenotypic diversity within the neonatal HSV-2 population

More than 14,000 neonates are infected with herpes simplex virus (HSV) annually. Approximately half display manifestations limited to the skin, eyes, or mouth (SEM disease). The rest develop invasive infections that spread to the central nervous system (CNS disease or encephalitis) or systemically (disseminated disease). Invasive HSV disease is associated with significant morbidity and mortality, but viral and host factors that predispose neonates to these forms are unknown. To define viral diversity within the infected neonatal population, we evaluated ten HSV-2 isolates from newborns with a range of clinical presentations. To assess viral fitness independent of host immune factors, we measured viral growth characteristics in cultured cells and found diverse in vitro phenotypes. Isolates from neonates with CNS disease were associated with larger plaque size and enhanced spread, with isolates from cerebrospinal fluid (CSF) exhibiting the most robust growth. We sequenced complete viral genomes of all ten neonatal viruses, providing new insights into HSV-2 genomic diversity in this clinical setting. We found extensive inter-host and intra-host genomic diversity throughout the viral genome, including amino acid differences in more than 90% of the viral proteome. The genes encoding glycoprotein G (gG, US4), gI (US7), gK (UL53), and viral proteins UL8, UL20, UL24, and US2 contained variants that were found in association with CNS isolates. Many of these viral proteins are known to contribute to cell spread and neurovirulence in mouse models of CNS disease. This study represents the first application of comparative pathogen genomics to neonatal HSV disease. ImportanceHerpes simplex virus (HSV) causes invasive disease in half of infected neonates, resulting in significant mortality and permanent cognitive morbidity. The factors that contribute to invasive disease are not understood. This study reveals diversity among HSV isolates from infected neonates, and makes the first associations between viral genetic variations and clinical disease manifestations. We found that viruses isolated from newborns with encephalitis show enhanced spread in culture. These viruses contain protein-coding variations not found in viruses causing non-invasive disease. Many of these variations are found in proteins known to impact neurovirulence and viral spread between cells. This work advances our understanding of HSV diversity in the neonatal population and how it may impact disease outcome.

microbiology↗